Fig 2: Usp16 and p16Ink4a modulates Rspo-mediated LRP6 phosphorylation. (a) MEFs cells were transiently transfected with siRNAs targeting Usp16 or p16Ink4a and treated with or without Wnt. Representative staining for βcatenin (in red) and DAPI (in blue) is shown on the left. On the right, the graph shows quantification of βcatenin intensity. Treatment with siRNA against Usp16 or p16Ink4a increases levels of βcatenin upon treatment with 50 ng/ml of Wnt3a. Experiments were repeated twice with two different MEF cultures. (b) Usp16+/− mammary epithelial cells, both untreated and treated with Wnt3a, express higher levels of Rspo1 and Rspo2 (n = 3 per group). (c) FACS-sorted mammary basal cells heterozygous for Usp16 express higher levels of Lgr5 upon treatment with Wnt3a (n = 4 per group). (d) Extracts derived from MEF cells and treated with the indicated siRNA were tested by western blot. Phospho-LRP6 immunoblots are shown on the left panel. βactin is used as loading control. A white line has been introduced between samples for clarity, but the image has not been cropped. On the right, the graph shows quantification of phospho-LRP6 intensity. Experiments were repeated four times.

Fig 3: Usp16 trisomy affects the Wnt pathway in Down’s Syndrome. (a) SYBR Green analyses revealed a marked decrease in the expression of a Top-Gal reporter of Wnt activity in mammary epithelial cells FACS-sorted from Ts65Dn mice compared to wt animals (n = 3 per group). (b) TTFs derived from animals with the indicated genotypes were analyzed for their ability to activate the Top-flash reporter in response to 20 ng/ml of Wnt3a. Data were normalized based on induction in wt animals. Wt and Ts65Dn/Usp16+/− cells activate Top-Flash more efficiently than Ts65Dn-derived cells. (wt, n = 5; Ts65Dn, n = 5; Ts65Dn/Usp16+/−, n = 2). (c) Microarray analysis shows clustering of wt, Ts65Dn and Ts65Dn/Usp16+/− RNA expression based on a Wnt signature of genes differentially expressed between wt and Ts65Dn cells (p < 0.05). (n = 2 per group). Ts65Dn/Usp16+/− RNA expression (samples in the middle) clusters with wt expression (samples on the right). (d) FACS-sorted epithelial cells from Ts65Dn mammary glands form less colonies in vitro compared to their wt counterpart. Axin2 heterozygosis increases the ability of Ts65Dn cells to form colonies (n = 3 animals per group; n = 2 for wt animals). Shown is passage P0. (e,f) Real time PCR analyses showed that human DS foreskin fibroblasts are less responsive to Wnt3a than cells derived from healthy patients in the activation of AXIN2, RSPO2 and RSPO3 (healthy, n = 4: DS, n = 3).

Fig 4: Heterozygosis of Usp16 in mammary tissue promotes Wnt-driven in vitro and in vivo cell expansion. (a) FACS analysis shows a higher basal to luminal cell ratio in the preneoplastic mammary gland of virgin MMTV-Wnt1-Usp16+/− mice. On the left, representative FACS plots of Lin− (Ter119− CD45− CD31−) mammary cells for the indicated genotypes. On the right, quantification of basal/luminal cell ratio. Each dot represents an individual mouse. (b–c) Histological analyses of preneoplastic mammary glands reveal an increase in the number and area of ducts derived from MMTV-Wnt1-Usp16+/− mice. The graph shows the average of six slides analyzed per animal, two animals per group. Quantification was performed with ImageJ software. On panel C, two representative pictures per genotype are shown. Keratin 8 and Keratin 14 were used to mark luminal and basal cell layers, respectively. The white bar scale is 100 μm. (d) FACS-sorted epithelial cells from MMTV-Wnt1-Usp16+/− preneoplastic mammary glands form more colonies in vitro compared to control mice (n = 3 per group). Shown is passage P1. (e) Usp16+/− sorted mammary epithelial cells show an increased induction of Axin2 mRNA levels 16 hours after Wnt3A stimulation (50 ng/ml). Three independent experiments were performed. (f) The mammary epithelial TCF-GFP+ frequency is increased in Usp16+/− compared to wt TCF-GFP animals after one passage in vitro. Each dot represents an individual animal. Unpaired T-test shows a two-tailed pvalue < 0.01. (g) Limiting dilution analysis (ELDA) shows an increased frequency of mammary repopulating cells in Usp16+/− compared to wt breast tissue. Four rounds of transplants were performed. The table shows the stem cell frequency and the 95% confidence interval between lower and upper values. (h) Usp16+/− cells were able to engraft better than wt cells in secondary recipients. Two independent experiments were performed. Each circle represents a recipient. In black, the estimated area occupied by the mammary tree in the fat pad two months after transplantation.

Fig 5: Cdkn2a mediates Usp16 response to Wnt3a. (a) Basal cell expansion is observed in the mammary gland of Cdkn2a+/− mice. A Graph quantifying the basal/luminal ratio is shown. Each dot represents an individual mouse. (b) Cdkn2a+/− sorted basal cells show an increased induction of Axin2 mRNA levels after 16 hours of Wnt3A stimulation (50 ng/ml). Two independent experiments were performed for a total of 4 animals per group. (c) Terminal Tip Fibroblasts (TTFs) derived from Cdkn2a+/− animals show an increase response to two different doses of Wnt3a (20 ng/ml and 50 ng/ml). The graph shows activation of a Top/Flash reporter for Wnt activity normalized by Renilla expression, co-transfected along with the reporter. (d) TTFs derived from Cdkn2a−/− animals and transfected with a vector expressing a Usp16 transgene or a control vector were analyzed for their ability to activate the Top-flash reporter. No differences were observed with two different doses of Wnt3a (20 ng/ml and 100 ng/ml). (e) MEFs cells were transiently transfected with two individual siRNAs targeting Bmi1. Activation of 6KD Wnt reporter is shown, normalized by Renilla expression. Cells were treated with 100 ng/ml of Wnt3a for 16 hours. (f) Two different human foreskin cultures were transiently transfected with siRNA targeting USP16, p16Ink4a or a scramble sequence. USP16 and p16Ink4a downregulation promotes the induction of AXIN2 mRNA expression 24 hours after treatment with 100 ng/ml of Wnt3a.